Toxoplasma gondii is a model system for genetic analyses in parasites belonging to the phylum Apicomplexa, a group that includes malaria and several additional human and animal pathogens. Like other apicomplexans, T. gondii has a complex life cycle although the underlying molecular bases of developmental transitions are poorly understood. Because the genome is relatively large (80 Mb) and contains many introns, EST sequencing is considerably more efficient than genomic sequencing for generating useful data about genes and their expression. (AIM1): To facilitate gene discovery in T. gondii, we will generate a comprehensive set of expression site tags (EST) designed to cover the majority of genes expressed by the three invasive stages in the life cycle: tachyzoites, bradyzoites, and sporozoites. Two of these stages, bradyzoites and sporozoites, are currently inadequately represented in the gene databases despite being important in transmission to and chronic infection of humans. By comparison to the existing gene databases, the expanded T. gondii ESTs will identify phylogenetically conserved genes and those unique to the Apicomplexa. They will also identify many strain-specific alleles for single-nucleotide polymorphism mapping. (AIM2): The ESTs generated here will be exploited for DNA microarray analyses of stage-specific, gene expression. DNA microarrays will be used to explore changes in gene expression during sporulation and during differentiation from tachyzoite to bradyzoite forms. In combination with the genetic tools available in T. gondii, this will greatly accelerate investigations into the complex developmental biology of apicomplexan parasites.